Registration Dossier

Administrative data

Endpoint:
genetic toxicity in vivo, other
Remarks:
Comet Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between10 October 2016 and 14 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was assigned Reliability 1 as the method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2014).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: GL500
Test item (alternative names): LGflex GL500
GL500
GL520
CAS No: 1571954-81-8
Action of test item: Plasticizer
Appearance/Physical state: Clear colorless liquid
Batch: GLFG160607
Purity: 99.8%
Expiry date: 07 June 2017
Storage conditions: Room temperature in the dark
Specific details on test material used for the study:
Identification: GL500
Test Item (alternative names): GL500, GL520
Purity: 99.8%
CAS No.: 1571954-81-8
Action of test item: Plasticizer
Batch number: GLFG160607
Storage Conditions: At ambient temperature (10 to 30 ºC) in the dark (although may be used and formulated in light).
Purity/weighing factor: No adjustment for purity was made
Appearance: Clear colourless liquid
Expiry date: 07 June 2017

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HsdRCCHan™WIST
Sex:
male
Details on test animals and environmental conditions:
Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo RMS (UK) Limited. At the start of the main test the males weighed 179.5 to 215.5 g, and were approximately eight to ten weeks old. Details of the individual animal weights, group means and standard deviations for the animals used in the main test are presented in Table 1. After a minimum acclimatization period of seven days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.

The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Arachis OIl
Details on exposure:
Experimental Design and Study Conduct

Test Item Preparation
For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil.
Determination by analysis of the concentration, homogeneity and stability of the test item preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.


Positive Control Preparation
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant Batch no. 3012083).

Vehicle Control
The Vehicle control (Arachis oil) was used as supplied.

Procedure
Range-finding Toxicity Test

A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 24 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

Groups of rats were dosed orally as follows:
Dose Level (mg/kg) Concentration (mg/mL) Dose Volume (mL/kg) Number of Rats
2000 200 10 2 male, 2 female

All animals were dosed twice 24 hours apart at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.
Animals were observed 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation.

Comet Test
Groups each of five males, were dosed twice with a 24 hour interval via the oral route with the test item at 2000, 1000 or 500 mg/kg. The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 4 hours following the second administration. In addition, two further groups of rats were included in the study; one group (five male rats) was dosed twice with a 24-hour interval via the oral route with the vehicle alone (arachis oil) and a second group (five male rats) was dosed twice orally with a 24-hour interval with N-Nitroso-N-methylurea (MNU) to act as the positive control. MNU is a positive control item that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test. The vehicle and positive control groups of rats were killed by humane euthanasia (carbon dioxide asphyxiation) 28 hours after the start of the test.

The experimental design is summarized as follows:

Treatment group Dose Level (mg/kg) Concentration (mg/mL) Dose volume (mL/kg) Kill Time (Hours After Initial Dosing) Animal Numbers
1. Vehicle Control (Arachis oil) 0 0 10 28 1 – 5
2. Positive Control (MNU) 25 2.5 10 28 6 – 10
3. GL500 2000 200 10 28 11 – 15
4. GL500 1000 100 10 28 16 – 20
5. GL500 500 50 10 28 20 – 25

All animals were observed for signs of overt toxicity and death one hour after each dosing and then immediately prior to termination.


Duration of treatment / exposure:
All animals were dosed twice 24 hours apart at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.
Animals were observed 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation.
Frequency of treatment:
See above
Doses / concentrationsopen allclose all
Dose / conc.:
500 other: mg/kg
Dose / conc.:
1 000 other: mg/kg
Dose / conc.:
2 000 other: mg/kg
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Identification: N-Nitroso-N-methylurea
Supplier: Astatec Inc
Batch Number: P102-01944
Purity: 90%
Expiry Date: 12 April 2017
Solvent: Water

Examinations

Tissues and cell types examined:
Tissue Sample Requirements

Humane euthanasia was performed on the animals at the end of the exposure period, using a method that did not affect the integrity of the required tissues (carbon monoxide asphyxiation). Samples of liver and glandular stomach were obtained from each animal.

Sub-samples of the liver and glandular stomach were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations. Assessment of cytotoxicity by histopathology is conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.

Details of tissue and slide preparation:
Tissue Preparation

Liver - A small piece of liver was excised (approximately 1 cm^3) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.

Glandular Stomach – The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by further scraping of the exposed tissue.

The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed as quickly as possible using ice-cold buffers to maintain the tissues and cell preparations at low temperature


Slide Preparation

Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature prior to the start of the experiment. Prior to use in the study the slides were labelled for animal number, project number and tissue type.

Once the cell suspensions had been obtained, approximately 30 µL was added to 270 µL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify. All of the slides went through the subsequent processing.

Once the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight.

After the lysis phase had been completed the slides were removed from the lysing solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis unit, which was filled with chilled electrophoresis buffer, until the slide surface was just covered. The slides were then left for approximately 20 minutes to allow the DNA to unwind. When the DNA unwinding period had finished the slides were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for approximately 20 minutes. The buffer in the bath was maintained at low temperature (approximately 2-10 °C) during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis. The voltage and current at the start and end of the electrophoresis period was recorded. The aim was to induce sufficient migration of the DNA so that minimal sized Comets are produced in the nuclei of vehicle control cells.

At the end of the electrophoresis period the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer (0.4M Tris pH 7.5) and allowed to rest for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer performed twice. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.

Once dry the slides were stored prior to scoring. Two of the four processed slide gels were scored and the remaining slides were stored as backup slides.
Evaluation criteria:
Acceptability Criteria

The following criteria will be used to determine a valid assay:

• The concurrent negative control is comparable with the laboratory historical negative control range.
• The positive controls induce responses that are comparable with those in the laboratory positive control range.
• Adequate numbers of cells and doses have been analysed.
• The highest dose level selected meets the requirements of the guideline and the study plan
Statistics:
When a less than clear response is observed a comparison will be made between the vehicle control groups and each corresponding treatment dose group on the percentage tail intensity data using individual slide scored values.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Range-Finding ToxicityTest

A range-finding test was performed to find suitable dose levels of the test item following oral administration at zero and 24 hours. The data are summarized as follows:

Dose Level (mg/kg)

 

Sex

Number of Animals Treated

 

Route

Deaths on Day

 

Total Deaths

0

1

2000

Male

2

oral

0

0

0/2

2000

Female

2

oral

0

0

0/2

In animals dosed with test item there were no premature deaths, and no clinical signs observed after dosing or at termination.

Based on the above data the maximum recommended dose (MRD) of the test item,2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg selected as the lower dose levels. There was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test. Due to the absence of any toxicity at the maximum recommended dose level the groups for the main test were limited to five animals per group.

Comet Assay

Mortality Data and ClinicalObservations

There were no premature deaths or clinical signs seen in any of the test item dose groups during the main test.

 

Evaluation of Comet AssaySlides

A summary of the results for each of the tissues of the Comet Assay, liver and glandular stomach, is given in Table 1.

The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

There was no marked increase in percentage tail intensity for any of the test item dose levels in the glandular stomach or liver tissues when compared to the vehicle control, confirming the test item did not induce DNA damage in the liver or glandular stomach.

There was no marked increase in hedgehog frequency for any of the test item dose levels in any of the tissues investigated.

Table 1            Summary of Results for Percentage Tail Intensity,MedianPercentageTail Intensity and Percentage Hedgehogs for EachTissue

 

Glandular Stomach 

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Mean of Median % Tail Intensity per Animal

Vehicle

5.27

7.41

5.22

500 mg/kg

6.55

6.37

4.03

1000 mg/kg

5.15

4.51

2.74

2000 mg/kg

6.04

4.99

3.28

Positive (MNU)

6.95

40.70

38.85

 

 

Liver 

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Mean of Median % Tail Intensity per Animal

Vehicle

0.68

0.37

0.01

500 mg/kg

0.30

0.35

0.01

1000 mg/kg

0.39

0.27

0

2000 mg/kg

0.40

0.35

0.01

Positive (MNU)

0.29

26.67

26.67

Applicant's summary and conclusion

Conclusions:
The test item did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach and therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo, under the conditions of the test.
Executive summary:

The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Ticeet al., 2000. The method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2014).

 

The primary target tissues of this assay were liver and glandular stomach.

 

Methods

 

A range-finding test was performed to find suitable dose levels of the test item and to determine the appropriate sex for the main test. The Comet assay main test was conducted at the maximum recommended dose (MRD) 2000 mg/kg with 1000 and 500 mg/kg as the lower dose levels, using five male animals per group. Animals were killed 4 hours after the second dose administration, the glandular stomach and liver tissues processed. Comet slides were then prepared and processed prior to scoring.

 

Further groups of rats were given a double oral dose of arachis oil (five rats) or N-Nitroso-N- methylurea (five rats), to serve as vehicle and positive controls respectively.

 

Results

 

The test item was tested up to the maximum recommended dose level of 2000 mg/kg using male animals only. There was no evidence of an increase in the percentage tail intensity or median percentage intensity in animals dosed with the test item dose groups when compared to the concurrent vehicle control group.

 

The positive control material produced a marked increase in the percentage tail intensity and median percentage tail intensity in both the liver and glandular stomach, indicating that the test method was working as expected.

 

Conclusion

 

The test item did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach and therefore the test item was considered to be unable to induce DNA strand breakage to these tissuesin vivo,under the conditions of the test.